At a Glance

A family history of anemia in the absence of iron deficiency should prompt consideration of a hemoglobinopathy, and a number of these appear in the differential diagnosis. Hemoglobin Köln is an inherited mutation in the ß-globin gene. It is predominantly confined to persons of German and Dutch descent.

Hemoglobin Köln is an unstable, high-affinity oxygen carrier that produces a chronic hemolytic condition. The peripheral smear of persons with Hemoglobin Köln trait (1 mutated gene) shows macrocytic hypochromic red blood cells (RBCs), with bite deformities and tear drop cells. Hemolysis is usually compensated by a reticulocytosis mounted in response to tissue hypoxia, so anemia is minimal or absent. The patient has scleral icterus and splenomegaly resultant from the chronic hemolysis. Occasionally, polycythemia is present (PCV > 55%) if the degree of hypoxia outweighs the hemolysis.

Family history reveals autosomal dominant inheritance pattern, but there are occasional de-novo mutations.

Homozygosity for hemoglobin Köln is incompatible with life.

Significant anemia or pronounced microcytosis (<65 fL) with hemoglobin Köln should prompt further investigations for the coinheritance of another hemoglobinopathy, such as a ß-thalassemia.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

If the Sickling test is positive, a variant hemoglobin is suspected from HPLC, or there is clinical suspicion of a hemoglobinopathy, isoelectric focusing (IEF) or electrophoresis (EP) of hemoglobin dimers (or less commonly free globin chains) should be ordered.

Screening tests for unstable hemoglobins could be ordered, but are only necessary if the diagnosis is uncertain from HPLC or IEF.

Assessment of iron status is important in anemia, which requires tests for ferritin and transferrin saturation (<20 ng/mL and <15%, respectively, in uncomplicated iron deficiency). Iron overload can occur with this condition and is associated with transferrin saturations greater than 75%.

Since there is macrocytosis, B12 and folate status should be checked and supplemented if deficient.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications - OTC drugs or Herbals - that might affect the lab results?

Hemoglobin Köln is the most common of the 130 or so unstable variant hemoglobins. An unstable hemoglobin should be suspected when chronic hemolytic anemia is associated with a Hgb trait, assuming there is no alternative explanation for the hemolysis (the most common of these are E, H, Köln, and Hasharon).

The substitution of methionine for valine at position 98 of the ß-globin chain (ß98 Val → Met) is charge-neutral, so it does not alter the electrophoretic properties of Hemoglobin Köln. However, this is an important site for both ß-ß-globin and heme binding, resulting in unstable tetramers and loss of heme. The latter results in loss of negative charge, and the chains become positive relative to Hgb A. If excess heme is added to the lysate, Hgb Köln migrates with Hgb A.

Denatured destabilized hemoglobin Köln attaches to the RBC membrane and impairs its deformity. During passage through the spleen, macrophages punch out these inflexible portions of the RBCs and significantly shorten their lifespan. These deposits form Heinz bodies, which, in Hgb Köln, are larger and more irregular than those seen with either α- or ß-thalassemias. They usually appear in the peripheral blood only after splenectomy, which is of little value in this disorder. In addition, the risk for thromboembolism secondary to a hypercoagulable state is increased by splenectomy.

The abnormally high oxygen affinity of hemoglobin Köln arises, because the mutation is in the FG corner of the globin chain, which is crucial to the normal movement of the α and ß chains that occurs during oxygenation/deoxygenation, trapping oxygen within the tetramer. This increased oxygen affinity produces a compensatory erythrocytosis, and, if it were not for the hemolysis that also occurs, the patient would have polycythemia.

Hemolysis is exacerbated by conditions that increase oxidant stress, which should be avoided. These include:

inherited G6PD deficiency

drugs reduced during their metabolism, such as dapsone, rifampicin, phenylhydrazines, phenazopyridine, and sulfur antibiotics

foods containing alkaloids that need reduction (e.g., fava beans)

infection

It is important to establish the true iron status of the patient before considering iron supplements or transfusion, as hemoglobinopathies with hemolytic anemias are iron loading. Anemia of inflammation (anemia of chronic disease) has a normal/elevated ferritin, and, in inflammatory disease/infection, C-Reactive Protein is elevated.

Transfused blood is always assumed to comprise 100% hemoglobin A, but this is not always the case as patients who are heterozygous for hemoglobin C or D mutations are not identified during donation, and this could alter the expected percentage of hemoglobin A and thus complicate the picture.

What Lab Results Are Absolutely Confirmatory?

The demonstration of substitution of methionine for valine at position 98 of the ß-globin chain is diagnostic for hemoglobin Köln (ß98 Val → Met). The expense of this test is rarely justified.

In practice, however, the demonstration of a broad hump of denatured hemoglobin prior to the normal Hgb A peak on HPLC, together with a broad smear from Hgb A through A2 on IEF (or a similar feature between Hgb S through C on alkaline EP and an additional band cathodal to Hgb A2 on acid EP), is considered confirmatory for the presence of hemoglobin Köln. Providing that the percentages of hemoglobin F and A2 are normal and the clinical severity is as expected, further testing is not usually warranted.

The percentage of Hemoglobin Köln is always significantly lower than that of hemoglobin A (typically only 25%), even though this variant is produced at a normal rate. Instability of the hemoglobin tetramer causes hemolysis.

Many Newborn Screening programs include tests for common hemoglobinopathies, but it is unlikely that hemoglobin Köln will be identified specifically. However, once the percentage of hemoglobin F is subtracted from the total hemoglobin, the same ratio of Köln to A will be observed as in adults.

A negative Sickling test should be observed with hemoglobin Köln, with the exception of splenectomized patients in whom the presence of large numbers of normoblast nuclei cause strong persistent turbidity. Splenectomy is not beneficial in hemoglobin Köln.

What Confirmatory Tests Should I Request for My Clinical Dx? In addition, what follow-up tests might be useful?

Sequencing of the chromosome for the known specific point mutations for ß-globin Köln may be indicated if the techniques previously mentioned are unable to arrive at a definitive diagnosis.

Sequencing of the chromosome for common point mutations or deletions in the ß-globin gene is only rarely indicated in the event of the appearance of a previously unknown hemoglobin entity.

If the severity of the clinical presentation does not match the initial diagnosis, sequencing of the α- and/or ß-globin transcription regulator genes, or sequencing of the gene in its entirety may be necessary to arrive at a definitive diagnosis. An elevated percentage of Hemoglobin A2 is indicative of a ß-thalassemia. If using the value of hemoglobin A2 as a key indicator of ß-thalassemia, it is crucial to exclude the presence of A2prime. This delta chain variant is clinically benign, but will be present at equal concentration to the A2, and must be added to it. It can be difficult to visualize on EP or IEF since the percentage is small. The rule of thumb that an MCV/RBC less than 14 is highly suggestive of ß-thalassemia is invalid in this condition. An elevated percentage of Hemoglobin F is suggestive of a ß0 thalassemia.

Determination of G6PD activity may be helpful, as inherited deficiency is common and will exacerbate hemolysis. Testing is readily available for G6PD activity, but this, ideally, should be assessed under baseline (no hemolysis present) conditions. Genetic testing for G6PD may, therefore, be more informative.

Treatment with the hydroxyurea increases the percentage of hemoglobin F present. Hydroxyurea is given for polycythemias, sickle disease, and as a chemotherapeutic agent.

What Factors, If Any, Might Affect the Confirmatory Lab Results? In particular, does your patient take any medications - OTC drugs or Herbals - that might affect the lab results?

The instability of Hemoglobin Köln on HPLC, IEF, and EP produces patterns that are difficult to interpret and may be attributed to poor sample preparation and, thus, be ignored as significant entities.

The current generation of hemoglobin A1C (glycated hemoglobin) assays have eliminated previously observed unreliability in the presence of hemoglobin C and S trait but may still give unreliable results with Hemoglobin Köln trait. Immunoassay-based tests perform better than HPLC, but results of diabetes tests should be interpreted with caution in these patients.

The Sickling test is a screening test that detects any hemoglobin that polymerizes under reduced oxygen tension and cannot differentiate between Homozygous S disease or one of the sickle traits or the presence of a doubly substituted S mutation, such as Hemoglobin C-Harlem. All results should be confirmed by additional testing, especially if they do not agree with the clinical picture.

The Sickling test may give a false negative if the Hemoglobin S concentration is below 1 g/dL (typically <10-15% of the total hemoglobin), after transfusion, or in cases where the F is greater than 90% (neonates and hereditary persistence of fetal hemoglobin.

The Sickling test may give a false positive if there are nucleated RBCs in the peripheral blood, as may occur after splenectomy or if the patient has a marked hypergammaglobulinemia, such as multiple myeloma.

There are many causes of hemolysis other than hemoglobinopathies, some of which are:

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